The present invention relates to fragments of HIV-1 Vpr protein which function in nuclear localization, virion packaging, cell cycle arrest and/or cell differentiation. The present invention relates to non-Vpr proteins which comprise such fragments of HIV-1 Vpr protein. The present invention also relates to methods of using such fragments and proteins to localize proteins to a cell""s nucleus, in assays to identify compounds which inhibit virion packaging, to method of arresting the cell cycle and to methods of inducing cell differentiation.
The present invention is related to U.S. Ser. No. 08/019,601 filed Feb. 19, 1993, issued as U.S. Pat. No. 5,874,225; U.S. Ser. No. 08/167,608 filed Dec. 15, 1993, now allowed; U.S. Ser. No. 08/246,177 filed May 19, 1994, issued as U.S. Pat. No. 5,639,598; U.S. Ser. No. 08/309,644 filed Sep. 21, 1994, issued as U.S. Pat. No. 5,763,190 on Jun. 9, 1998; U.S. Ser. No. 08/809,186 filed Feb. 3, 1995, issued as U.S. Pat. No. 5,780,220; U.S. Ser. No. 08/505,196 filed Oct. 11, 1995, now abandoned; and U.S. Ser. No. 08/809,186 filed Mar. 20, 1997, issued as U.S. Pat. No. 6,667,157; which are each incorporated herein by reference.
The human immunodeficiency virus type 1 (HIV-1) accessory gene vpr, while dispensable for viral replication in T-cell lines and activated primary peripheral blood mononuclear cells (PBMC), is required for efficient replication in primary monocyte/macrophages.
The Vpr protein has been characterized as an oligomer. HIV-2 and SIV code for a second protein, Vpx, which shares considerable sequence homology with. Both proteins are packaged efficiently in HIV and SIV viral particles. Virion localization studies place both Vpr and Vpx outside the core structure. Although Vpr and Vpx are not part of the Gag structural polyprotein, their incorporation requires an anchor to associate with the assembling capsid structures. The C-terminal portion of the Gag precursor corresponding to the p6 protein appears to constitute such an anchor through an unknown mechanism. In addition, p6 is essential for the incorporation of both Vpr and Vpx into virus particles. A predicted putative xcex1-helical domain near the amino terminus plays an important role in the packaging of Vpr into virions and in maintaining protein stability.
Several possible roles have been suggested for Vpr in HWV-1 replication. Vpr can modestly transactivate HIV-1 LTR and thus may upregulate viral gene expression in newly infected cells before the appearance of Tat. It has been found to enhance the nuclear migration of the preintegration complex in newly infected nondividing cells. Significantly, Vpr induces cellular differentiation which includes the activation of specific host cell gene transcription and growth arrest in several tumor cell lines, even in the absence of any other viral proteins. This suggests that Vpr, itself, may be sufficient to alter cellular functions. Vpr has been reported to block cell cycling in G2/M phase of the cell cycles. This finding has been associated with a change in the phosphorylation state of CDC2 kinase. Furthermore, Vpr expression appears to inhibit the establishment of chronic infection. Vpr has been reported to causes growth arrest and structural defects in yeast. Functional studies have shown that Vpr accelerates HIV-1 replication in some T-lymphoid cells lines in primary macrophages where the effects of Vpr are more pronounced.
It has also been reported that Vpr has transcellular activity. Both Vpr purified from plasma of HIV-1 seropositive individuals and purified recombinant Vpr, were capable of inducing latent cells into high-level viral producer when added to culture media at low concentrations. Mechanistically, it is conceivable that this transcellular activity is mediated by the same mechanisms which modify cellular growth and differentiation. It has been reported that Vpr is primarily localized in the nucleus when expressed in the absence of other HIV-1 proteins. Although, no classical nuclear localization signal has been clearly identified for Vpr, it has been suggested that Vpr may gain access to the nucleus by specific interactions with nuclear proteins. In this regard, proteins that interact with Vpr in host cells have been reported but not molecularly cloned. Interestingly, one of these Vpr targets, designated Vpr-interacting protein, or RIP-1, appears to be translocated to the nucleus following its interaction with Vpr or triggering by glucocorticoid receptor (GR) ligands, supporting a possible role for the GR pathway in Vpr function.
The molecular relationship between these different functions of Vpr have not yet been defined. There is a need to identify the functional activity of the various domains of Vpr protein.
The present invention relates to conjugated compositions comprising a fragment of HTV-1 Vpr comprising amino acid sequence 17-36 and/or 59-84 or a non-HIV-1 Vpr protein comprising amino acids amino acids 17-36 and 59-84 conjugated to a therapeutic compound.
The present invention further relates to methods of delivering a therapeutic compound to the nucleus of a cell. The method comprises the step of contacting the cell with a conjugated compound that is either the therapeutic compound conjugated to a fragment of HIV-1 Vpr protein comprising amino acids 17-36 and/or 59-84 or the therapeutic compound conjugated to a non-HIV-1 Vpr protein comprising amino acids 17-36 and/or 59-84 of HIV-1 Vpr protein. The conjugated compound is taken up by said cell and localized to the nucleus of the cell.
The present invention further relates to fragments of HIV-1 Vpr comprising amino acid sequences 17-36 and/or 59-84 and to non-HIV-1 Vpr proteins comprising amino acids 17-36 and/or 59-84 of HIV-1 Vpr protein.
The present invention further relates to methods of inhibiting cell proliferation. The methods comprise the step of arresting said cell""s advance in the cell cycle by contacting said cell with a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein; or a nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein. The fragment of HIV-1 Vpr or non-HIV-1 Vpr protein is taken up by said cell or the nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein or the nucleic acid molecule that encodes the non-HIV-1 Vpr protein is taken up by the cell and expressed to produce the fragment of HIV-1 Vpr or non-HIV-1 Vpr protein in the cell. The fragment of HIV-1 Vpr or non-HIV-1 Vpr protein inhibits said cell from advancing in said cell cycle.
The present invention further relates to methods of treating an individual who has a hyperproliferative disease. The method comprises the step of administering to the individual in an amount effective to inhibit cell proliferation a composition comprising: a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein; or a nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein. The fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule is taken up by proliferating cells of the individual or the nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein or the nucleic acid molecule that encodes the non-HIV-1 Vpr protein is taken up by a proliferating cell of the individual and expressed to produce the fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule in the cell. The fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule inhibit the cell from advancing in the cell cycle.
The present invention further relates to a fragment of HIV-Vpr comprising amino acid sequence or a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein.
The present invention further relates to pharmaceutical composition comprising a fragment of HIV-Vpr comprising amino acid sequence or a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-81of HIV-1 Vpr protein and a pharmaceutically acceptable carrier.
The present invention further relates to a nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein; and to pharmaceutical compositions comprising the same.
The present invention further relates to methods of identifying compounds that inhibit Vpr protein binding to the p6 domain of p55 or to p6 protein. The methods comprise the steps of contacting a fragment of HIV-1 Vpr comprising amino acid sequence 17-36 or a non-HIV-1 Vpr protein comprising amino acids 17-36 of HIV-1 Vpr protein with a protein comprising an HIV-1 Gag protein p6 domain in the presence of a test compound. The level of binding between the fragment of HIV-1 Vpr or the non-HIV-1 Vpr protein and the protein comprising an HIV-1 Gag p6 domain is determined and compared to the level of binding between the fragment of HIV-1 Vpr or the non-HIV-1 Vpr protein and the protein comprising an HIV-1 Gag p6 domain contacted in the absence of a test compound.
The present invention further relates to kits for identifying compounds which inhibit Vpr protein binding to p55""s p6 domain or to p6 protein. The kits comprise a first container comprising a fragment of HIV-1 Vpr comprising amino acid sequence 17-36 or a non-HIV-1 Vpr protein comprising amino acids 17-36 of HIV-1 Vpr protein; and a second container comprising a protein comprising an HIV-1 Gag protein p6 domain.
The present invention further relates to fusion proteins comprising Vpr amino acid sequence 17-36 and non-Vpr amino acid sequences, and drug delivery particles comprising the same.